[advances in chemistry] hydrolysis of cellulose: mechanisms of enzymatic and acid catalysis volume...

2 The Hydrolysis of Cellulosic Materials to Useful Products

ARTHUR E. HUMPHREY

University of Pennsylvania, Philadelphia, PA 19104

Because cellulose is the most abundant renewable resource, it should have great promise as a source of liquid fuel, food, and chemical feedstocks. Based on present technology, cellulose utilization through hydrolysis processes does not appear economical for the production of sugar syrups or alcohol fuels, particularly if biomass costs are greater than $30/ton. However, there is reason for optimism. If ways can be found to improve yields, specifically to achieve total biomass utilization and to improve the value of the process by-products, then process feasibility may emerge. Two important costs factors are raw materials costs and pretreatment methods to improve yields and biomass utilization. Also, new ways of squeezing the water from the resulting cellulose fermentation products could enhance the process economics.

/Cellulose is the most abundant renewable resource available for con-^ version to fuel, food, and chemical feedstocks. It has been estimated by Ghose (11 ) that the annual worldwide production of cellulose through photosynthesis may approach 100 Χ 109 metric tons. As much as 25% of this could be made readily available for the conversion processes. A significant fraction of the available cellulose, i.e., 4-5 Χ 109 t/year, occurs as waste, principally as agricultural and municipal wastes. Cellulose must be viewed, therefore, as an important future source of fuel, food and chemicals (see Table I).

Cellulose hydrolysis and its product, glucose, play a central role in the conversion of renewable resources to foods, fuel, and chemical feedstocks. This is illustrated in Figure 1. Cheap glucose would not only find a demand in the food sweetener market but could serve as a substrate

0-8412-0460-8/79/33-181-025$07.25/0 © 1979 American Chemical Society

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In Hydrolysis of Cellulose: Mechanisms of Enzymatic and Acid Catalysis; Brown, R., et al.; Advances in Chemistry; American Chemical Society: Washington, DC, 1979.

26 HYDROLYSIS OF CELLULOSE

Table I. Supply and Demands for the Year 2000

Demand

liquid fuels food chemical feedstocks

total

109 t/year

10 6

_1 17

Supply

potentially available cellulose 25

for the production of fuel, alcohol, and single-cell protein. There is a greater choice of organisms that can grow on glucose compared with other substrates. Also, with glucose substrates there should be less problem with undesirable or toxic residues when using them to produce single-cell protein.

With respect to the hydrolysis step, it can be accomplished by acid, by enzymatic, or by direct microbial attack. Microbial hydrolysis results primarily in the production of cellular biomass or single-cell protein. Acid hydrolysis, while simple and direct, results in a sugar syrup with considerable contamination from the side reaction products. Enzymatic hydrolysis is usually the cleanest hydrolysis process. Unfortunately, it is the most costly of the three to operate.

7 v X photosynthes is

sol id combustion fuel

p l a s t i c s

l ignin - hydrolysis processes

wastes

hemicel lu lose pr imari ly x y l o s e

fodder yeast

food sugar y through ^ isomer izat ion

g lucose single ce l l

v. protein ^ ^ t h r o u g h

fermentat ion \ ^ food

x y l I t o l

fodder

a lcoho l through fermentat ion

pro te in - g l y c a n s -nucleic ac id

c h e m i c a l s -ethylene through A dehydration

liquid fue ls g a s o h o l

Figure 1. The centrality of cellulose hydrolysis in the conversion processes

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2. H U M P H R E Y Feedstock Production 27

The Nature of Cellulose

For the most part, cellulosic hydrolysis studies have been concerned with pure or at least relatively pure cellulose. However, cellulose in its natural state never occurs in a pure form. It always is associated with lignin and hemicellulose. The portions are indicated in Table II.

Table II. Composition of Cellulosic Materials

Material % of Dry Matter

cellulose 40-60 hemicellulose 20-40 lignin 10-25

The hemicellulose fraction is a mixture of sugars, primarily pentoses. Depending upon the biomass source, the hemicellulose fraction can be as much as 85% xylan and yields xylose on hydrolysis. Xylose can be used by many microorganisms either fermenting to alcohols or converting to microbial biomass. Xylose can also be hydrogenated to xylitol, a potentially important diabetic sweetener. The lignin, although only on the order of one-fifth of the total biomass, represents approximately 50% of the available combustible energy in naturally occurring sources of cellulose. It seems obvious, therefore, that any cellulose hydrolysis scheme must be prepared to utilize both the lignin and hemicellulose.

Acid Hydrolysis Process

Acid hydrolysis of cellulosic materials has been studied for many years (13,33). Although it is a relatively straightforward process, it has the problems of requiring acid-resistant equipment and yielding a poor grade of sugar (because the product contains many reaction product impurities). However, in terms of practical application, acid hydrolysis of cellulosic material is by far the most commonly used hydrolysis system.

A two-stage acid hydrolysis process is employed in over 40 Soviet wood hydrolysis plants (15). These plants have an average annual output per plant of 10,000 t of wood sugar. Most of the output is converted to industrial alcohol and fodder yeast.

The major soluble components of acid hydrolysates are sugars, such as xylose, glucose, and cellobiose; furfurals, such as furfuraldehyde and hydroxymethyl furfural; and organic acids, such as levulinic acid, formic acid, and acetic acid (13). When natural sources of cellulose are acid-hydrolized, numerous products can result, largely because of the hemicellulose materials. These make it difficult to produce a relatively pure sugar product and limit the utility of the acid hydrolysis process.

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In order to minimize the product contamination, the acid hydrolysis process is usually performed in two stages. The first stage involves contact with dilute acid (1% sulfuric) at relatively low temperatures (80-120°C) and short times (30-240 min). The purpose of this stage is to extract the hemicellulose fraction, mostly as pentoses. The second stage is performed with stronger acid (from 5-20% sulfuric) and higher temperatures (approximately 180°C) . The purpose of the second stage is to hydrolyze the cellulose to glucose. The overall objective is to optimize conditions such that the glucose yield is maximized and the secondary product contamination is minimized.

Cellulosic materials are quite variable from source to source, not only in cellulose, hemicellulose, and lignin content but also in the crystallinity of the cellulose. As a consequence, each natural substrate would be expected to have its own unique set of process conditions to optimize glucose yield and minimize secondary product contamination. The next section on kinetics of acid hydrolysis will examine this point.

Acid Hydrolysis Kinetics

A considerable amount of experimentation has been done on the kinetics of acid hydrolysis of pure cellulose substrates. Little experimentation has been done on natural cellulosic materials. Typical examples of kinetic studies of acid hydrolysis of cellulose can be found in the papers of Saeman (33) and Grethlein (13). These researchers depict the acid hydrolysis process as a pseudo-first-order sequential process, with the rate constants as a function of the acid concentration raised to a power, i.e.,

cellulose > glucose (C.) (Ci)

(1)

where

dC. di fci C, X (2)

k2 d (3)

(4)

and where

fct — j K i W e x p i - . E i / B T ) (5)

fc2 =K2{A)nexp{-E2/RT) (6)

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In these expressions, Cx = concentration of cellulose, C i = concentration of glucose, C0 = concentration of decomposed glucose products, kx and k2 are the rate constants for the respective reactions, Yi and Y2 are the stoichiometric coefficients, and Λ is the acid concentration.

Grethlein (13) and Saeman (33) have estimated values of the various constants for acid hydrolysis of Solka Floe and Douglas Fir. These are given in Table III.

Table III. Acid Hydrolysis Constants for Various Cellulosic Materials

Constant Douglas Fir Solka Floe

Kl9 m i n 1 1.73 Χ 1019 1.22 Χ 1019

K2, min1 2.38 Χ 1014 3.79 Χ 1014

El9 cal/g · mol 42,900 42,500 E2, cal/g · mol 32,800 32,700 m 1.34 1.16 η 1.02 0.69

The fact that these constants are quite similar for Douglas Fir and Solka Floe is remarkable since both behave quite differently when enzy-matically hydrolyzed. It would be expected that each cellulosic material would behave differently. For most cellulosic materials, there is a unique temperature at a fixed acid concentration and reaction time that gives optimal glucose yield. This is illustrated in Figure 2, which summarizes the glucose yield data of Grethlein (13) for acid hydrolysis of Solka Floe as a function of temperature for a fixed residence time (0.22 min) at various acid concentrations (0.5, 1.0, 1.5, and 2% sulfuric acid) and solids concentrations (5-13.5% cellulose). Since 0.22 min approaches a practical minimal contact time that can be achieved, due to mass-transfer lags in economically sized cellulosic substrates, it is doubtful that greater than 50-60% conversion of cellulose to glucose is practically realizable without appreciable secondary product formation. More work is needed on short-time, high-temperature, and high-concentration acid hydrolysis. It would appear that acid hydrolysis, because of the glucose breakdown products, will be limited in its applicability. Conversion of the glucose syrup to fuel ethanol is the only use envisioned at the present time.

Enzyme Hydrolysis Process

There are many sources of cellulolytic enzymes; however, the fungus Trichoderma viride has proved to be the most effective source to date. The microbiology (25,32) and enzyme kinetics (24,30) studies on this organism have been pioneered by workers such as Reese and Mandels at

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30 HYDROLYSIS O F C E L L U L O S E

TIME, hrs.

Figure 3. Typical enzymatic digestion of cellulose

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the U.S. Army Natick Laboratories. With proper pretreatment, such as ball milling, a cellulase preparation from T. viride is able in a reasonable time ( < 100 hr) to break down completely pure cellulose substrates. This is not true for natural sources of cellulose such as bagasse or wood chips. With these substrates, it is usually difficult to obtain much more than 50% conversion. The problem is that natural sources of cellulose are protected from enzymatic hydrolysis by associated hemicelluloses and lignin. Either the removal of lignin and hemicelluloses from the native cellulose or the addition of hemicellulases is necessary to improve the glucose yield in enzymatic hydrolysis of naturally occurring cellulosic materials.

In a typical enzymatic hydrolysis of a 5% suspension of ball-milled newsprint, a sugar syrup containing 1.6% glucose, 1.4% cellobiose, and 0.2% xylose is readily obtained. Figure 3 is illustrative of kinetic results obtained with T. viride cellulase at 3.5-filter-paper-units/mL ( FP units/ mL) strength in a 5% suspension of ball-milled newsprint.

There are two reasons for the measurable cellobiose concentration in the T. viride cellulase hydrolyzed syrups. The most likely is that T. viride has rather poor β-glucosidase activity so that cellobiose accumulates. Evidence of this is that additions of β-glucosidase to the T. viride cellulase improves its activity. A second reason is that the β-glucosidase enzyme is strongly glucose inhibited. Hence the rate of cellobiose hydrolysis slows down as the glucose concentration rises, allowing cellobiose to accumulate.

At least three major cellulase components are involved in cellulose hydrolysis (6,8,19,39). These are endo-β-glucanases, exo-β-glucanases, and β-glucosidase ( cellobiase ). The most widely accepted model for the enzymatic hydrolysis of pure cellulose is depicted in Figure 4. Crystalline cellulose (Cx) is attacked by endo-β-glucanases to give amorphous cellulose and some oligosaccharides. These materials are attacked in turn by exo-/?-glucanases to give glucose (Ci) directly and by the cellobiosyl-hydrolases to give cellobiose (C 2 ) , which, in turn, is hydrolyzed by β-glucosidase to give glucose. The latter is thought to be the dominant mechanism by which T. viride cellulases produce glucose. Consequently, the overall reaction may be depicted as a two-step reaction of cellulose going to cellobiose and then to glucose. Both steps are inhibited by their products.

The problem of modeling the hydrolysis kinetics is complicated by the fact that cellulose is a solid substrate; consequently, the reaction can be surface limited (5,12). Furthermore, some sites are more susceptible to hydrolysis—e.g., the amorphous regions as well as specific regions of the crystalline cellulose such as edges, corners, and dislocations. Several investigators (17,20,36) have suggested that the kinetic model should be based on a "shrinking site model" in which the number of susceptible

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sites is proportional to the surface area. Further, the enzyme must first be adsorbed at these sites in order to function. Therefore, the kinetic model should include an adsorption isotherm to relate the soluble enzyme concentration (Ex) to the adsorbed enzyme concentration (Eads) (4). An example of such a kinetic model is outlined in the next section.

Shrinking Site Hydrolysis Model

In evolving the shrinking site model, it will be assumed that the disappearance of cellulose can be represented by the following mechanisms:

h h

Cx >C2 >d (7)

where Cx is the solid cellulose substrate concentration and C 2 and C i are the cellobiose and glucose concentrations. The disappearance of cellulose can then be given by

dCx - Γ h Ί ' x l h + c2_ where

Κ = reaction rate constant -Eads == grams of absorbed enzyme/gram of cellulose

I2 = cellobiose inhibition concentration, g/L

If one assumes that a Langmuir-type adsorption isotherm can be used to relate the concentration of adsorbed cellulase on the cellulose to the free cellulase in solution, i.e.,

_« + Ex_ Eaas = E°ads Ι ι π (9)

and

E\dB = K'v4*rR2n (10) where

E°&dB = saturation concentration of cellulase/gram of cellulose Kf = no. adsorption sites/gram of cellulose

η = no. adsorption sites/unit surface area JB = mean mass radius/cellulose particle n = no. cellulose particles/gram of cellulose

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and noting that

= A VR*PN (11)

or

R = (SCJlnpN)1* Ρ = grams/milliliter of cellulose

Ν = no. cellulose particles/liter solution

then

da dt

--^[-^rhhi] 1131

Using Equation 13 as a starting point, it is now possible to construct a kinetic model for the enzymatic hydrolysis of cellulose. This model is summarized in Table IV. Six kinetic parameters are required to define the system. With the proper selection of parameter values, the data depicted in Figure 3 can be duplicated by the model. Wilkie and Yang (37) have suggested that a simple distribution coefficient is adequate for expressing the adsorption isotherm of Cx and C x activity on a — 20-mesh Wiley-milled newsprint (see Figure 5). These data were obtained at a relatively low enzyme activity where a linear isotherm would be expected to apply. The distribution coefficient was very small (D = 0.04 FP units/mL/FP units/g solids), which suggests that the cellulase enzyme binds rather tightly to the solid cellulose. It also suggests that simple countercurrent

Table IV. Enzymatic Cellulose Hydrolysis

h h ι Ι ι I Ex I E2 I

Cx > C2 > C\

Cellulose: - -W [^][^]

„ „ .. dC2 . dCx Γ K2E2C2 Ί Γ h Ί Cellobiose: = + - ^ - ^ J ^ - ^ j

Glucose: = + ^-^^—^-J

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100^ \ \

V

DISTRIBUTION COEFFICIENT c l \ X = 0 .0MFP u n i t s / m l ) / ( F P u n i t s /

gm solids)

J I L 2.5 5.0 7.5 1 0

NEWSPRINT CONCENTRATION gm/100ml

Figure 5. Data of Wilke and Yang on adsorption of C1 and C x cellulase activity (in terms of FP units) on —20-mesh Wiley-milled newsprint.

Distribution coefficient = 0.04 (FP units/mh)l(FP units/g solids).

adsorption of the cellulase from the sugar syrup exiting from the hydro-lyzer will permit a high degree of cellulase recovery and reuse.

Moreira in his PhD thesis work attempted to verify the shrinking site model by estimating the adsorbed protein on the solid cellulose as a function of cellulose digestion and time (28). The data suggest that a Langmuir-type adsorption equilibrium occurs and that as the cellulose hydrolysis occurs, a condition is reached where all the adsorption sites are saturated. Then, as the cellulose hydrolysis proceeds, adsorbing sites disappear and enzyme is ejected form the shrinking cellulose surface and goes back into solution.

Microbial Hydrolysis of Cellulose

Microbial hydrolysis of cellulose can often be very direct, fast, and complete. Even though a microorganism may produce primarily an extracellular cellulase, hydrolysis is usually faster in the presence of organisms than just the cellulase-containing solution alone. The reason for this is that cellulolytic organisms grow on the cellobiose or glucose, thus continuously removing them from solution and relieving their inhibitory effects. This high hydrolysis rate is illustrated in Figure 6, where data for the growth of a Thermoactinomyces sp. at 60°C on Avicel is given (2). Note that the maximum rate of cellulose hydrolysis, occurring at

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2. HUMPHREY Feedstock Production 37

8-10 hr into the fermentation, is equivalent to a volumetric cellulose hydrolysis rate of nearly 2 g/L-h. This kind of volumetric rate is considered excellent for most fermentative processes.

In modeling a microbial process for the hydrolysis of cellulose, one must account for the generation of the enzymes as the organism grows and the repression of the enzyme production due to glucose. One model that has appeared in the literature (20) for this process is given in Table V. This model is viewed as a rather simplistic expression of what actually

Table V. Cellulose Fermentation: Shrinking Site Model (Microorganism grows only on glucose)

Ce„ulose: *k _ -K,C.« [-^-J^]

CellobioSe: *k_+^-[^][7_^_]

Cells: = + *n" °lX

dt 1 Ks + d

Enzyme,: ^f- = ΥΕχ/Σ (l -

„ dE2 v (Λ Ci_\dX Enzyme,: = ΥΕ2/Σ - - ^ - ^ j ^ -

Typical Values for Parameters

Kx = 0.084 (g/L) a = 0.30 g/L h - 1.0 g/L κ2

= 10.0 g/L-hr KM = 4.0 g/L h = 1.0 g/L Y = 0.5 g/g

m = 0.01g/g-hr Mmax = 0.25 L/hr K, = 0.1 g/L YEX/X = 0.01 g/g

0.03 g/g 0.01 g/L

= 0.01 g/L

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occurs. Even such a simplistic model has 14 constants or parameters that must be evaluated. Parameter identification of this number of constants for a single process is most difficult. However, the model does predict the behavior of a typical batch fermentation of cellulose (see Figure 7). The model clearly indicates that relatively little benefit is obtained from microbial cellulose digestion beyond 50% conversion unless the cellulose costs are high and one is concerned with the cellulose-to-cell protein ratio and wants to minimize this ratio in the final product.

The model in Table V assumes that the microbial cells excrete an extracellular endoglucanase, which is adsorbed on the solid cellulose and initiates the hydrolysis process, which ends in glucose that is taken up by the cells. The work of Binder and Ghose (3) suggest that this may not be so in some cases of cell growth on cellulose. For example, they found T. viride cells had essentially a constant adsorption-distribution coefficient for cotton cellulose and could adsorb several times their own weight of cellulose (3,4) (see Figure 8). They found the process of adsorption of cellulose on cells to be rather slow. In contrast, the adsorption process for soluble enzyme on solid cellulose substrate is relatively fast. Therefore, the model depicted in Table V is believed to be more consistent with observed results than the cell-cellulose adsorption model proposed by Binder and Ghose (3).

2.5 5 7.5 1.0

X ( m g K j e l d a h l ni trogen)

Figure 8. Data of Binder and Ghose on adsorption of powdered cotton cellulose by cells of T. viride. Note: T. viride is approximately 8.0% Kjeldahl nitrogen on a dry basis. Therefore, the T. viride cells are capable

of adsorbing 9.4 times their weight of cellulose.

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In studies of cellulose utilization by some species of cellulolytic organisms, it has been observed that growth on cellulose occurs at a faster rate than that on glucose (16,21). Also, when cellobiose is used as the substrate, it has been noted that glucose accumulates in appreciable quantities (2). Various mechanisms have been offered to explain this phenomenon. However, it is now known that many cellulolytic organisms prefer to grow on cellobiose. Indeed, they have the capability of phos-phorylating cellobiose and taking up this organic molecule directly without first having to hydrolyze it. Production of glucose is a secondary reaction. Only when cellobiose has been fully utilized will the organism turn to utilizing the glucose. Figure 9 is illustrative of the typical kind of data one gets when such an organism is grown on cellobiose. Note the manner in which glucose accumulates and then is utilized. Such behavior is similar to diauxic growth.

Table VI presents a kinetic model for preferred growth of a microorganism on cellobiose (C 2 ) but having an extracellular β-glucosidase (E2) that is glucose inhibited and in which the glucose uptake is repressed. This model readily fits the cellobiose data shown by the solid lines in Figure 9 (20).

This behavior is one explanation of why organisms can grow so well on pretreated or ball-milled cellulose. The growth rates are only limited by the number of active sites or surface area available to them for attack per unit concentration of cell biomass or enzyme. An organism (X) will grow at a near-maximum growth rate until such time that the maintenance demand of that organism (m) is not being met by the rate of carbonaceous energy supply. Growth then begins to slow down and finally stops when the energy supply is less than the maintenance demand, i.e.,

and

J ^ L = 1 . ^ + mX (14) at Yx/cx dt

as long as

When

Yx/cx dt

d C * > mX dt

dCx

dt = mX

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H U M P H B E Y Feedstock Production 41

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Table VI. Growth of Tbermoactinomyces on Cellobiose

C2 > X

Cells :

dX _ /*max2 C2X ι / -ι C2 λ /»ma*i ClX dt - KS2 + C2 R2 + C2) K8I + C1

Glucose: dd _ . Γ K2E2C2 ΊΓ 7t Π _ / C2 \ ftmaxx CiX

dt ^IKM + Csjlh + Cx] \L R2 + C2) K3L + C1

Enzyme 2 :

dE2 dX dt - Ϊ Ε 2 / Χ ~dT

Table VII. Growth Parameters for Tbermoactinomyces on Various Substrates

m Y (g substrate/ (g cell/

Substrate pmax (hr'1) g cell · hr) g substrate)

Cellulose 0.4-0.5 0.02-0.08 0.40-0.5 Cellobiose 0.2 0.027 0.27 Glucose 0.48 0.038 0.44 Oxygen/glucose — 0.032 1.03

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growth ceases, and when

-lif<~x

growth ceases and/or cell lysis occurs. Typical growth parameters for a Thermoactinomyces sp. on cellulose,

cellobiose, and glucose are given in Table VII. This table suggests that cellulose hydrolysis must occur at a rate greater than 0.02 to 0.04 g cellulose/g organism-hr for cell growth to occur. These data further illustrate just how important pretreatment of the cellulosic material is to good growth and yield of microorganisms on cellulose.

Practical Cellulose Hydrolysis Processes

Both pilot-plant and plant-scale processes for cellulose hydrolysis or digestion by acids, enzymes, and microorganisms have been built (1, 7,15,27). Acid and enzyme processes usually have as their objective the production of a sugar syrup, while the microbial process usually results in microbial protein for animal feed. Figure 10 is illustrative of a microbial process (29) that has been developed to convert the unused cellulosic material in manure to recycle feed. Similar processes have been developed

WASH

S L A T T E D PENS

SAND ETC.

R E C Y C L E COLLOIDAL PROTEIN

DRIED R E C Y C L E

FEED

Figure 10. Animal recycle feedlot wastes system

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for waste straw and for cane bagasse. As previously indicated, the problem is to achieve an adequate yet cheap cellulose pretreatment process. To date, simple grinding has been inadequate, drying and ball milling is too expensive, and combined alkali and heat treatment is marginally economical (26). Just recently, steam explosion of dried substrates and solvent delignification have been suggested as pretreatment procedures. It is the opinion of this author that the major bottlenecks to economical cellulose hydrolysis processes are the availability of large supplies of cheap biomass, the cost of the pretreatment step, and the conversion yield of the total biomass, i.e., hemicellulose as well as cellulose.

Figure 11 gives a scheme proposed by Wilke and Yang (27,37) for the enzymatic hydrolysis of cellulose. The enzymatic process has a number of additional economic bottlenecks. These involve the cost of production of the cellulase enzyme and the recovery or reuse of enzyme. In order to achieve a high enzyme reuse, it is necessary to recover the enzyme from the glucose syrup product by adsorbing the enzyme on fresh cellulosic material. Similarly, it is necessary to recover the enzyme from spent cellulosic solids by washing. Both recovery systems are essential to economical enzymatic processes. Fortunately, the various cellulase components have similar adsorption characteristics (see Figure 5), thus their relative effectiveness is not significantly changed or reduced (37).

Figure 11. Cellulose hydrolysis process

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The economic potential of enzyme hydrolysis is therefore dependent upon the evolution of ( 1 ) cheap, readily available, large biomass sources, (2) cheap and efficient pretreatment methods, and (3) cheap and potent sources of cellulolytic enzymes.

The data in Figure 12 highlight the effect of substrate costs on the cost of glucose for a 250,000-t/yr plant. Most people think that large sources of naturally occurring biomass (50-60% cellulose) will only be available at a delivered price of $30 per ton O D W (oven-dried weight) (18). If this is true, glucose syrups will only be produced by the enzymatic process at costs, depending upon the percentage of cellulose utilization, of 8 to 160 per pound of sugar. Such processes are marginally attractive at best in the present economic price climate for world sugar.

Conversion of Sugar to Alcohol and Other Products

The conversion of sugar to alcohol has been highly touted as a way of solving the fuels and chemical feedstock problem. Clearly, ethyl alcohol is a technically feasible alternative to gasoline and ethylene for chemical feedstocks. The question is whether it is a practical or economic solution. Various economic exercises have been made (7,16,22,23,34, 37,38). Some are favorable; others are highly unfavorable. The key is determining the basic assumptions; in particular, what is the cost of the raw material? The sensitivity of the process economics to raw material costs is illustrated in Figure 12. Another problem is by-products credits, i.e., the process cost reduction due to the by-products such as animal feed, spent solid fuel, recovered polymer grade lignin, etc. This is illustrated in Figure 13.

The other problem is the process energy demand due to "squeezing the water out" of the fermented alcohol product stream in order to produce high-purity alcohol. Unfortunately, most fermentations, such as alcohol fermentation, are carried out in dilute aqueous solution. Considerable energy is required to separate the alcohol from the water by steam distillation. These energy costs, in terms of tons of oil for energy per ton of alcohol product at various alcohol product concentrations, are illustrated in Figure 14. When oil costs were low, the energy costs of the separation process were relatively minor to the overall costs. Today, they are greater than 40% of the total process costs.

Examination of the data in Figure 14 indicates that the alcohol process should produce at least an 8% and preferably a 10-14% ethanol feed stream from the fermentation process in order to minimize separation costs. This means, assuming a 40% ethanol weight yield based on glucose in the syrup, that at least a 20-25% sugar syrup must be produced for fermentation. Without an intermediate sugar concentra-

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20r

LU t/> Ο Ο 3

10 LU ο C L

LU to

DATA FOR 250,000 T/YR P L A N T 90° /o O V E R A L L P R O C E S S E F F I C I E N C Y 50°/o E N Z Y M E RECOVERY ( R E - U S E )

P R O B A B L E DEL IVERY COST OF C E L L U L O S E FOR B IOMASS A V A I L A B L E IN L A R G E VOLUMES

10 20 30 AO 50

$ / T O N OF D E L I V E R E D C E L L U L O S E

60

Figure 12. Relation of glucose selling price to delivered cellulose substrate costs for enzymatic hydrolysis process. Data for 250,000 t/yr plant:

90% overall process efficiency, 50% enzyme recovery (reuse).

tion step, the enzymatic hydrolysis process must be run on very high solids substrates. Indeed, solid bed reactors may be the only feasible reactor configuration for hydrolysis systems.

The ethanol selling price is very sensitive to the glucose substrate costs. This is illustrated in Figure 15. Present hydrolysis technology suggests that glucose substrates can be produced in the lO-to-140-per-pound range. Unfortunately, this results in costs of $1.60 to $2.00 per gallon for alcohol. In order to compete with (1978) U.S. gasoline prices, alcohol must sell for 60 to 800 per gallon. This requires a 2-to-40-per-pound glucose feed (Figure 14), which in turn means that cellulosic biomass must be available at a delivered price of less than $10/t of cellulose content. It would seem, therefore, that until the price of gasoline rises to the $1.60-per-gallon range, the use of ethanol produced from biomass to augment gasoline and other fuels is not economically attractive. However, there are places in the world where gasoline sells at this level and where large quantities of cheap biomass are readily available. An alcohol-based fuel economy may be feasible for these locations. Further, with decontrol of oil prices, gasoline prices of this level may soon be reached.

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to

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2.00

1.00

ο ζ < χ PRESENT TECHNOLOGY CAPABILITY

4 6 θ 10 GLUCOSE SUBSTRATE COSTS IN φ/ lb

Figure 15. Relation of ethanol selling price to glucose substrate costs

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Total Biomass Utilization Concept

In all the economic considerations, only the cellulosic portion of the biomass has been considered. Recently, several investigators have pointed out that by-product credits can change the process economics drastically. With simple economics, such as presented here, they have argued that

01 I I I I I L

0 1 2 3 4 5 6

ELAPSED TIME (hours)

Figure 17. Hydrolysis of poplar chips by extracellular cellulose of Ther-moactinomyces YX. (25 dry g chips/L. Chips wet-milled in blender be

fore hydrolysis.)

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"total biomass utilization' and by-product credits must be taken into account in order to give cellulose hydrolysis processes any practical standing.

One such proposed "total biomass utilization" scheme is presented in Figure 16 (31). The basic concept is to solvent-delignify the biomass in order to recover a valuable lignin product, either for polymer or diesel fuel use, and to utilize the hemicelluloses for alcohol fermentation. Preliminary data suggest that solvent-delignified biomass can be very susceptible to enzymatic hydrolysis (see Figure 17 (31) ). These preliminary results are most encouraging and give optimism to those searching for ways to develop economical hydrolysis processes.

Other developments have occurred that give additional optimism and hope in achieving an economical alcohol process from hydrolyzed cellulose. One such development is the combined saccharification and fermentation technique with saccharifying and alcohol-producing organisms (35), i.e., yeast or CI. thermocellums. Another involves the suggestion that the water or dilute product problem might be overcome by high-temperature (60°C) vacuum or solvent-extractive fermentation (9). Here, the alcohol concentration relative to the water content of the product could be enhanced in a rather simple, one-step, low-energy-demand process.

Summary

Based on present technology, cellulose hydrolysis processes do not appear to be economical for the production of sugar syrups or alcohol fuels, particularly if biomass costs are greater than $30/t. However, there is reason for optimism. Most important in contributing to this optimism is the emerging concept of total biomass utilization and the possibility of process by-product credits. Further, cheaper and more potent cellu-lases are being developed. Cheaper pretreatment methods that give greater cellulose utilization are emerging. Finally, new fermentation techniques are being evolved that minimize the problem of "squeezing the water" out of the fermentation product.

Literature Cited

1. Andren, R. K. "Proc. Symp. on Bioconversion of Cellulosic Substances into Energy, Chemicals and Microbial Protein," New Delhi, Feb 1977, pp. 397-412.

2. Armiger, W. B.; Moreira, A. R.; Phillips, J. Α.; Humphrey, A. E. Pac. Chem. Eng. Congr. [Proc.] 1977, 1, 196.

3. Binder, Α.; Ghose, T. K. Biotechnol. Bioeng. 1978, XX, 1187. 4. Bisaria, V. S.; Ghose, T. K. "Proc. Symp. on Bioconversion of Cellulosic

Substance into Energy, Chemicals and Microbial Protein," New Delhi, Feb 1977, pp. 155-164.

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5. Brown, D. E.; Waliuzzaman, M. "Proc. Symp. on Bioconversion of Cellulosic Substances into Energy, Chemicals and Microbial Protein," New Delhi, Feb 1977, pp. 351-371.

6. Emert, G. H. ; Brown, R. D. "Proc. Symp. on Bioconversion of Cellulosic Substances into Energy, Chemicals and Microbial Protein," New Delhi, Feb 1977, pp. 153-155.

7. Emert, G. H.; Huff, G. Special Senate Hearing on Alcohol Fuels, Jan. 31, 1978.

8. Fagerstam, Lars; Hakansson, U.; Pettersson, G.; Andersson, L. "Proc. Symp. on Bioconversion of Cellulosic Substances into Energy, Chemicals and Microbial Protein," New Delhi, Feb 1977, pp. 165-178.

9. Finn, R. K. Biotechnol. Bioeng. Symp. 1975, 5, 353. 10. Gauss, W. F., et al., "Manufacture of Alcohol from Cellulosic Materials

Using Plural Ferments," U.S. Patent 3 990 944, assigned to the Bio Research Center Co., Tokyo, Japan, 1976.

11. Ghose, T. K. Adv. Biochem. Eng. 1977, 6, 39. 12. Ghose, T. K.; Das, K. Adv. Biochem Eng. 1971, 1, 55. 13. Grethlein, H. E. Biotechnol. Bioeng. Symp. 1975, 5, 503. 14. Huff, G. F.; Toda, Ν. Y. "Enzymatic Hydrolysis of Cellulose," U.S. Patent

3 990 945, assigned to the Bio Research Center Co., Tokyo, Japan, 1976. 15. Humphrey, A. E. "Single Cell Proteins: A Case Study in the Utilization of

Technology in the Soviet System," In "Soviet Science and Technology: Domestic and Foreign Prospectives"; Thomas,J. R., Kruse-Vaucienne, U. M., Eds.; George Washington University: Washington, DC, 1976; pp. 358-373.

16. Humphrey, A. E. "Symp. on Enzymatic Hydrolysis of Cellulose," Aulanko, Finland, March 1975, pp. 437-453.

17. Humphrey, A. E.; Moreira, E. R.; Armiger, W. B.; Zabriskie, D. Biotechnol. Bioeng. Symp. 1977, 7, 45.

18. Inman, R. E. Biotechnol. Bioeng. Symp. 1975, 5, 67. 19. King, K. W.; Vessal, M. I. Adv. Chem. Ser. 1969, 95, 391. 20. Lee, S. E. Ph.D. Dissertation, University of Pennsylvania, 1977. 21. Lee, S. E.; Armiger, W. B.; Watteeuw, C. M.; Humphrey, A. E. Biotech

nol. Bioeng. 1978, XX, 1, 141. 22. Linko, M. Adv. Biochem Eng. 1977, 5, 27. 23. Lipinsky, E. "Economics of Liquid Fuel from Corn Stover," Battelle

Report, 1978. 24. Mandels, M.; Hontz, L.; Nystrom, J. Biotechnol. Bioeng. 1974, 16, 1471. 25. Mandels, M.; Reese, Ε. T. J. Bacteriol. 1957, 73, 269. 26. Millett, Μ. Α.; Baker, A. J.; Satter, L. D. Biotechnol. Bioeng. Symp. 1975,

5, 193. 27. Mitra, G.; Wilke, C. R. Biotechnol. Bioeng. 1975, 17, 1. 28. Moreira, A. R.; Phillips, J. Α.; Humphrey, Α. Ε. "A Method for Deter

mining the Concentration of Adsorbed Protein and Cell Biomass in Cellulose Fermentations," submitted for publication in Biotechnol. Bioeng.

29. Nolan, E. J.; Shull, J. J. "Engineering Experiences During the Demonstration Phase of a Nutrient Reclamation Plant," 75th National AIChE Meeting, Detroit, June 1973.

30. Peitersen, N.; Medeiros, J.; Mandels, M. Biotechnol. Bioeng. 1977, 19, 1091.

31. Pye, Ε. K.; Humphrey, A. E., Progress Report on "The Biological Production of Organic Solvents from Cellulosic Wastes," Dept. of Energy Contract No. EY-76-S-02-4070, Report C0014070-6, Distribution Category UC-61, July 1978.

32. Reese, Ε. T. Biotechnol. Bioeng. Symp. 1972, 3, 43. 33. Saeman, J. Ind. Eng. Chem. 1945, 37, 43.

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34. Scheller, W. A. "The Production of Ethanol by the Fermentation of Grain," paper presented at the Intl. Symp. on Alcohol Fuel Technology, Wolfs-burg, Germany, Nov 1977.

35. Takagi, M.; Abe, S.; Suzuki, S.; Emert, G. H.; Yata, N. "Proc. Symp. on Bioconversion of Cellulosic Substances into Energy, Chemicals, and Microbial Protein," New Delhi, Feb 1977, pp. 551-571.

36. Van Dyke, Β. H., Jr. "Enzymatic Hydrolysis of Cellulose—A Kinetic Study," Ph.D. Dissertation, M.I.T., 1975, p. 328.

37. Wilke, C. R.; Yang, R. D. "Symp. on Enzymatic Hydrolysis of Cellulose," Aulanko, Finland, March 1975, pp. 485-506.

38. Wilke, C. R.; Mitra, G. Biotechnol. Bioeng. Symp. 1975, 5, 253. 39. Wood, T. M. Biotechnol. Bioeng. Symp. 1975, 5, 111. R E C E I V E D August 1, 1978.

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